Probiotic lactic acid bacteria and their biofilms have antagonistic activity against food spoilage organisms and pathogenic bacteria. Recently, researchers focused on the use of probiotic biofilms for inhibition of pathogenic bacteria. The aim of this research is to improve probiotic biofilms with optimal prebiotic concentration and to determine their inactivation effect on both planktonic cells and biofilm growth of Listeria monocytogenes. Biofilm formations were detected by using microplate method. Prebiotic ingredients were used to form biofilm with highest viable probiotic cell counts and optimal concentrations of prebiotic ingredients were determined according to the response surface method. Biofilm produced by Lactobacillus casei Shirota and Lactobacillus rhamnosus contained 9.46 and 9.66 log cfu/mL viable cell counts, respectively. Optimal prebiotic concentrations were found 3% casein peptone-0% fructo-oligosaccharides (FOS) for biofilm formation with highest viable cell counts by L. casei Shirota and 1.5% casein peptone-1.5% FOS for biofilm formation with highest viable cell counts by L. rhamnosus. Probiotic biofilms exhibited inactivation against growth of L. monocytogenes and caused a reduction of 0.66-2.01 log cfu/mL for planktonic L. monocytogenes and 0.40-1.69 log cfu/mL for L. monocytogenes biofilm. Planktonic cells of L. monocytogenes were observed to be more susceptible to probiotic biofilms than biofilm of L. monocytogenes. Biofilm of L. rhamnosus showed higher inhibition effect on L. monocytogenes growth than L. casei Shirota. These findings showed that biofilms of probiotic Lactobacillus strains used in this study may be excellent candidate for controlling of pathogenic bacteria.